BackgroundThe recently described Type VI Secretion System (T6SS) represents a new paradigm of protein secretion in bacteria. A number of bioinformatic studies have been conducted to identify T6SS gene clusters in the available bacterial genome sequences. According to these studies, Salmonella harbors a unique T6SS encoded in the Salmonella Pathogenicity Island 6 (SPI-6). Since these studies only considered few Salmonella genomes, the present work aimed to identify novel T6SS loci by in silico analysis of every genome sequence of Salmonella available.ResultsThe analysis of sequencing data from 44 completed or in progress Salmonella genome projects allowed the identification of 3 novel T6SS loci. These clusters are located in differentially-distributed genomic islands we designated SPI-19, SPI-20 and SPI-21, respectively. SPI-19 was identified in a subset of S. enterica serotypes including Dublin, Weltevreden, Agona, Gallinarum and Enteritidis. In the later, an internal deletion eliminated most of the island. On the other hand, SPI-20 and SPI-21 were restricted to S. enterica subspecies arizonae (IIIa) serotype 62:z4,z23:-. Remarkably, SPI-21 encodes a VgrG protein containing a C-terminal extension similar to S-type pyocins of Pseudomonas aeruginosa. This is not only the first evolved VgrG described in Salmonella, but also the first evolved VgrG including a pyocin domain described so far in the literature. In addition, the data indicate that SPI-6 T6SS is widely distributed in S. enterica and absent in serotypes Enteritidis, Gallinarum, Agona, Javiana, Paratyphi B, Virchow, IIIa 62:z4,z23:- and IIIb 61:1,v:1,5,(7). Interestingly, while some serotypes harbor multiple T6SS (Dublin, Weltvreden and IIIa 62:z4,z23:-) others do not encode for any (Enteritidis, Paratyphi B, Javiana, Virchow and IIIb 61:1,v:1,5,(7)). Comparative and phylogenetic analyses indicate that the 4 T6SS loci in Salmonella have a distinct evolutionary history. Finally, we identified an orphan Hcp-like protein containing the Hcp/COG3157 domain linked to a C-terminal extension. We propose to designate this and related proteins as "evolved Hcps".ConclusionAltogether, our data suggest that (i) the Salmonella T6SS loci were acquired by independent lateral transfer events and (ii) evolved to contribute in the adaptation of the serotypes to different lifestyles and environments, including animal hosts. Notably, the presence of an evolved VgrG protein related to pyocins suggests a novel role for T6SS in bacterial killing. Future studies on the roles of the identified T6SS loci will expand our knowledge on Salmonella pathogenesis and host specificity.

The genus Salmonella contains two species, S. bongori and S. enterica. Compared to the well-studied S. enterica there is a marked lack of information regarding the genetic makeup and diversity of S. bongori. S. bongori has been found predominantly associated with cold-blooded animals, but it can infect humans. To define the phylogeny of this species, and compare it to S. enterica, we have sequenced 28 isolates representing most of the known diversity of S. bongori. This cross-species analysis allowed us to confidently differentiate ancestral functions from those acquired following speciation, which include both metabolic and virulence-associated capacities. We show that, although S. bongori inherited a basic set of Salmonella common virulence functions, it has subsequently elaborated on this in a different direction to S. enterica. It is an established feature of S. enterica evolution that the acquisition of the type III secretion systems (T3SS-1 and T3SS-2) has been followed by the sequential acquisition of genes encoding secreted targets, termed effectors proteins. We show that this is also true of S. bongori, which has acquired an array of novel effector proteins (sboA-L). All but two of these effectors have no significant S. enterica homologues and instead are highly similar to those found in enteropathogenic Escherichia coli (EPEC). Remarkably, SboH is found to be a chimeric effector protein, encoded by a fusion of the T3SS-1 effector gene sopA and a gene highly similar to the EPEC effector nleH from enteropathogenic E. coli. We demonstrate that representatives of these new effectors are translocated and that SboH, similarly to NleH, blocks intrinsic apoptotic pathways while being targeted to the mitochondria by the SopA part of the fusion. This work suggests that S. bongori has inherited the ancestral Salmonella virulence gene set, but has adapted by incorporating virulence determinants that resemble those employed by EPEC.

We constructed two collections of targeted single gene deletion (SGD) mutants and two collections of targeted multi-gene deletion (MGD) mutants in Salmonella enterica sv Typhimurium 14028s. The SGD mutant collections contain (1), 3517 mutants in which a single gene is replaced by a cassette containing a kanamycin resistance (KanR) gene oriented in the sense direction (SGD-K), and (2), 3376 mutants with a chloramphenicol resistance gene (CamR) oriented in the antisense direction (SGD-C). A combined total of 3773 individual genes were deleted across these SGD collections. The MGD collections contain mutants bearing deletions of contiguous regions of three or more genes and include (3), 198 mutants spanning 2543 genes replaced by a KanR cassette (MGD-K), and (4), 251 mutants spanning 2799 genes replaced by a CamR cassette (MGD-C). Overall, 3476 genes were deleted in at least one MGD collection. The collections with different antibiotic markers permit construction of all viable combinations of mutants in the same background. Together, the libraries allow hierarchical screening of MGDs for different phenotypic followed by screening of SGDs within the target MGD regions. The mutants of these collections are stored at BEI Resources (www.beiresources.org) and publicly available.

Vibrio parahaemolyticus is the most common cause of seafood-borne gastroenteritis worldwide and a blight on global aquaculture. This organism requires a horizontally acquired type III secretion system (T3SS2) to infect the small intestine, but knowledge of additional factors that underlie V. parahaemolyticus pathogenicity is limited. We used transposon-insertion sequencing to screen for genes that contribute to viability of V. parahaemolyticus in vitro and in the mammalian intestine. Our analysis enumerated and controlled for the host infection bottleneck, enabling robust assessment of genetic contributions to in vivo fitness. We identified genes that contribute to V. parahaemolyticus colonization of the intestine independent of known virulence mechanisms in addition to uncharacterized components of T3SS2. Our study revealed that toxR, an ancestral locus in Vibrio species, is required for V. parahaemolyticus fitness in vivo and for induction of T3SS2 gene expression. The regulatory mechanism by which V. parahaemolyticus ToxR activates expression of T3SS2 resembles Vibrio cholerae ToxR regulation of distinct virulence elements acquired via lateral gene transfer. Thus, disparate horizontally acquired virulence systems have been placed under the control of this ancestral transcription factor across independently evolved human pathogens.Vibrio parahaemolyticus | transposon-insertion sequencing | type III secretion | bacterial pathogenesis | pathogen evolution T he gram-negative γ-proteobacterium Vibrio parahaemolyticus thrives in either pathogenic or symbiotic association with marine organisms and as a planktonic bacterium (1). This facultative human pathogen, abundant in aquatic environments, was first isolated following a food poisoning outbreak in 1952 and has emerged as the leading cause of seafood-associated gastroenteritis worldwide and a blight on global aquaculture (2, 3). Sequencing of the V. parahaemolyticus genome and the development of animal models of infection have demonstrated a critical role for type III secretion in V. parahaemolyticus virulence (4, 5).Pathogenic isolates of V. parahaemolyticus encode two type III secretion systems (T3SSs), which are multiprotein structures that mediate the translocation of bacterial effector proteins directly into eukaryotic cells (4, 6). All V. parahaemolyticus strains encode a T3SS on the large chromosome (T3SS1), and the vast majority of clinical isolates, but few environmental isolates possess a horizontally acquired pathogenicity island (VPaI-7) encoding a second T3SS (T3SS2) and one or more pore-forming toxins (TDH) (7). Studies using the infant rabbit model of V. parahaemolyticus infection, which recapitulates manifestations of human gastrointestinal disease (e.g., profuse diarrhea, enteritis, epithelial disruption), revealed that although T3SS1 and TDH are dispensable for intestinal colonization and pathogenesis, colonization and pathology are dependent on T3SS2, consistent with the epidemiological association between T3SS2 and pathogenicity (5). Furthermor...

The amount of lipopolysaccharide (LPS) O antigen (OAg) and its chain length distribution are important factors that protect bacteria from serum complement. Salmonella enterica serovar Typhi produces LPS with long chain length distribution (L-OAg) controlled by the wzz gene, whereas serovar Typhimurium produces LPS with two OAg chain lengths: an L-OAg controlled by Wzz ST and a very long (VL) OAg determined by Wzz fepE . This study shows that serovar Enteritidis also has a bimodal OAg distribution with two preferred OAg chain lengths similar to serovar Typhimurium. It was reported previously that OAg production by S. Typhi increases at the late exponential and stationary phases of growth. The results of this study demonstrate that increased amounts of L-OAg produced by S. Typhi grown to stationary phase confer higher levels of bacterial resistance to human serum. Production of OAg by serovars Typhimurium and Enteritidis was also under growth-phase-dependent regulation; however, while the total amount of OAg increased during growth, the VL-OAg distribution remained constant. The VL-OAg distribution was primarily responsible for complement resistance, protecting the non-typhoidal serovars from the lytic action of serum irrespective of the growth phase. As a result, the non-typhoidal species were significantly more resistant than S. Typhi to human serum. When S. Typhi was transformed with a multicopy plasmid containing the S. Typhimurium wzz fepE gene, resistance to serum increased to levels comparable to the non-typhoidal serovars. In contrast to the relevant role for high-molecular-mass OAg molecules, the presence of Vi antigen did not contribute to serum resistance of clinical isolates of serovar Typhi.

Salmonella enterica serovar Enteritidis causes a systemic, typhoid-like infection in newly hatched poultry and mice. In the present study, a library of 54,000 transposon mutants of S. Enteritidis phage type 4 (PT4) strain P125109 was screened for mutants deficient in the in vivo colonization of the BALB/c mouse model using a microarray-based negative-selection screening. Mutants in genes known to contribute to systemic infection (e.g., Salmonella pathogenicity island 2 [SPI-2], aro, rfa, rfb, phoP, and phoQ) and enteric infection (e.g., SPI-1 and SPI-5) in this and other Salmonella serovars displayed colonization defects in our assay. In addition, a strong attenuation was observed for mutants in genes and genomic islands that are not present in S. Typhimurium or in most other Salmonella serovars. These genes include a type I restriction/modification system (SEN4290 to SEN4292), the peg fimbrial operon (SEN2144A to SEN2145B), a putative pathogenicity island (SEN1970 to SEN1999), and a type VI secretion system remnant SEN1001, encoding a hypothetical protein containing a lysin motif (LysM) domain associated with peptidoglycan binding. Proliferation defects for mutants in these individual genes and in exemplar genes for each of these clusters were confirmed in competitive infections with wild-type S. Enteritidis. A ⌬SEN1001 mutant was defective for survival within RAW264.7 murine macrophages in vitro. Complementation assays directly linked the SEN1001 gene to phenotypes observed in vivo and in vitro. The genes identified here may perform novel virulence functions not characterized in previous Salmonella models.

SUMMARY Type III secretion systems (T3SSs) inject bacterial effector proteins into host cells and underlie the virulence of many Gram-negative pathogens. Studies have illuminated bacterial factors required for T3SS function, but the required host processes remain largely undefined. We coupled CRISPR/Cas9 genome editing technology with the cytotoxicity of two Vibrio parahaemolyticus T3SSs (T3SS1 and T3SS2) to identify human genome disruptions conferring resistance to T3SS-dependent cytotoxicity. We identity non-overlapping genes required for T3SS1- and T3SS2-mediated cytotoxicity. Genetic ablation of cell surface sulfation reduces bacterial adhesion and thereby alters the kinetics of T3SS1-mediated cytotoxicity. Cell surface fucosylation is required for T3SS2-dependent killing, and pharmacological or genetic inhibition of fucosylation prevents membrane insertion of the T3SS2 translocon complex. These findings reveal the importance of ubiquitous surface modifications for T3SS function, potentially explaining the broad tropism of V. parahaemolyticus, as well as highlight the utility of genome-wide CRISPR/Cas9 screens to discover processes underlying host-pathogen interactions.

Salmonella Gallinarum is a pathogen with a host range specific to poultry, while Salmonella Enteritidis is a broad host range pathogen that colonizes poultry sub-clinically but is a leading cause of gastrointestinal salmonellosis in humans and many other species. Despite recent advances in our understanding of the complex interplay between Salmonella and their hosts, the molecular basis of host range restriction and unique pathobiology of Gallinarum remain largely unknown. Type VI Secretion System (T6SS) represents a new paradigm of protein secretion that is critical for the pathogenesis of many Gram-negative bacteria. We recently identified a putative T6SS in the Salmonella Pathogenicity Island 19 (SPI-19) of Gallinarum. In Enteritidis, SPI-19 is a degenerate element that has lost most of the T6SS functions encoded in the island. In this work, we studied the contribution of SPI-19 to the colonization of Salmonella Gallinarum strain 287/91 in chickens. Non-polar deletion mutants of SPI-19 and the clpV gene, an essential T6SS component, colonized the ileum, ceca, liver and spleen of White Leghorn chicks poorly compared to the wild-type strain after oral inoculation. Return of SPI-19 to the ΔSPI-19 mutant, using VEX-Capture, complemented this colonization defect. In contrast, transfer of SPI-19 from Gallinarum to Enteritidis resulted in transient increase in the colonization of the ileum, liver and spleen at day 1 post-infection, but at days 3 and 5 post-infection a strong colonization defect of the gut and internal organs of the experimentally infected chickens was observed. Our data indicate that SPI-19 and the T6SS encoded in this region contribute to the colonization of the gastrointestinal tract and internal organs of chickens by Salmonella Gallinarum and suggest that degradation of SPI-19 T6SS in Salmonella Enteritidis conferred an advantage in colonization of the avian host.